Peptides for targeting gastric cancer, and medical use tehreof

ABSTRACT

Provided is a peptide for targeting gastric cancer, a composition for diagnosing radioresponsiveness-dependent gastric cancer using the peptide, and a drug delivery use of the peptide, wherein a functional peptide capable of targeting cancer has been discovered so as to implement personalized diagnosis and treatment for individual patients having cancer, consideration of problems occurring during treatment in which treatment cases of respective patients differ due to different therapeutic responses resulting from genetic differences in the individual patients.

TECHNICAL FIELD

The present invention relates to a peptide for targeting gastric cancer,a composition for diagnosing gastric cancer based on performance ofirradiation using the peptide, and drug delivery use of the peptide.

BACKGROUND ART

Cells which are the smallest unit of the human body maintain the balanceof cell number by cell division upon intracellular regulatory functions,cell growth, and cell death and disappear, when cells are normal. If thecells are damaged by any cause, cells may be recovered by treatment tothereby serve as normal cells. However, if cells are not recovered,cells die by themselves. A condition in which abnormal cells that do notcontrol proliferation and inhibition thereof for a variety of reasonsare excessively proliferated and also cause tumefaction and destructionof normal tissues by invading surrounding tissues and organs is definedas cancer. As such, cancer refers to cell proliferation that is notinhibited, and cancer destroys the structure and function of normalcells and organs. In this regard, it is significantly important todiagnose and treat cancer.

However, there are problems during treatment in which treatment cases ofrespective patients differ due to different therapeutic responsesresulting from genetic differences in the individual patients havingcancer. Thus, in order to effectively treat cancer patients, it isrequired to develop a functional targeting agent capable of targetingtumor microenvironment, which depends on radioresponsiveness, and abiomarker. Accordingly, it is possible to establish personalizeddiagnosis and treatment for individual patients.

In addition, drug delivery systems or targeted therapies thatselectively deliver drugs to cancer cells and cancer tissues aretechnologies that have received much attention, because even if the sameamount of an anticancer agent is used, drug efficacy may be increasedwhile side effects of drugs on normal tissues may be significantlyreduced at the same time. In addition, when such technologies areapplied to gene therapy, selective delivery of virus to cancer cells canincrease treatment efficacy and reduce severe side effects. For thispurpose, antigens that are mainly specific to tumor cells and antibodiesthat target such antigens have been developed up to date. However, inthe case of antibodies, there are problems including concerns of immuneresponse and low efficiency of penetration into tissues. In the case ofpeptides, a molecular weight thereof is so small that there is lessconcern of an immune responses and the penetration of peptides intotissues is easy. Therefore, if cancer-targeting peptides are coupledwith existing anticancer drugs, such resulting products can be utilizedas intelligent drug vehicles that selectively deliver drugs to tumors.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present invention, unlike screening methods that have been studiedat the existing cell culture levels, establishes mouse modelstransplanted with a cancer tissue of an actual human, to thereby dividethem into an irradiated population and a non-irradiated population as acontrol group. In addition, a method of screening a peptide thatspecifically binds to each population above is disclosed to provide anovel peptide for targeting gastric cancer and a medical use of such anovel peptide.

Technical Solution

To solve the technical problem above, the present invention provides apeptide for targeting gastric cancer and a polynucleotide encoding thepeptide, the peptide including an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1 to 6.

In an embodiment, the present invention provides a peptide for targetinggastric cancer, the peptide including an amino acid selected from thegroup consisting of SEQ ID NOs: 1 to 3.

In an embodiment, the present invention provides a composition includingthe peptide for diagnosing gastric cancer and a composition includingthe peptide for diagnosing radio-reactive gastric cancer.

the present invention provides a composition including the peptide fordelivering a drug.

Advantageous Effects of the Invention

present invention relates to a peptide for targeting gastric, acomposition for diagnosing radioresponsiveness-dependent gastric cancerusing the peptide, and a drug delivery use of the peptide. Consideringproblems during treatment in which treatment cases of respectivepatients differ due to different therapeutic responses resulting fromgenetic differences in the individual patients having cancer, afunctional peptide capable of targeting cancer has been discovered so asto establish personalized diagnosis and treatment for individualpatients. Animal models similar to cancer microenvironments of actualpatients having cancer are prepared and divided into irradiatedpopulations and non-irradiated populations as a control group, tothereby test target efficiency for respective peptides that are selectedby screening peptides specifically binding to the respectivepopulations. As such, the present invention can be finally utilized inthe technical development of image diagnosis for predictingresponsiveness to radiotherapy, and accordingly, the development ofcustomized targeted therapeutic agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image showing a method of establishing an irradiated animalmodel after transplanting an actual patient's gastric cancer tissue intoa mouse according to Example 1 of the present invention, and alsoshowing confirmation results of the established animal model. FIG. 1Ashows a patient's gastric cancer tissue distributed from the BioResearch Center (BRC, Korea), FIG. 1B shows a NOD/SCID mouse thatundergoes heterotrophic transplantation into the flanks of the mouse,FIG. 1C shows a cancer tissue cut into pieces each having a size of 3×3mm to be used for subculturing, when the size of the cancer tissue ofFIG. 1B is increased up to 500 mm³, FIG. 1D shows a mouse model preparedin a way that a nude mouse is anesthetized via intraperitoneal injectionand undergoes heterotrophic transplantation of one piece of the cuttissues subcutaneously on the both thighs, and FIG. 1E shows irradiationof 10 grays (Gy) of radiation over the thigh portions where the cancercell is formed, when the size of the cancer tissue is increased up to150-200 mm³. Here, a control group is not subjected to irradiation.

FIG. 2 shows a biopanning scheme for identifying a sequence of apeptide, which targets a gastric cancer tissue of an irradiated in vivopatient, by using an M13 phage display method according to embodimentsof the present invention.

FIG. 3 shows results of comparing phage concentrations obtained byeluting phages after extracting heart, lung, liver, spleen, kidney, andtumor during biopanning process performed five times.

FIG. 4 shows results of a linking proportion between a Cy5.5 fluorescentprobe and a phage by calculating phages of the same concentration aftera discovered peptide-expressing phage of the present invention isamplified in terms of linking with the Cy5.5 fluorescent probe, and bycalculating region of interest (ROI) values in connection with linkingbetween the Cy5.5 fluorescent probe and the phage.

FIG. 5 shows results confirming specific binding to in vivo gastriccancer tissue based on images on the 2^(nd) day after injecting apeptide phage labeled with a Cy5.5 fluorescent probe into each mousemode.

FIG. 6 shows results confirming fluorescence intensity of cancer tissueafter only cancer tissue is extracted and also confirming places wherefluorescence is located on cancer tissue that is equally divided, as invivo image confirmation is completed on the 2^(nd) day.

FIG. 7 shows a schematic diagram for observing changes in targetingability of a peptide phage of the present invention as being selecteddepending on irradiation. In detail, after a patient's gastric cancertissue is transplanted, mouse models in which the size of the cancertissue is increased up to 150-200 mm³ are divided into 1) irradiatedmouse models and 2) irradiated mouse models with 2 grays (Gy) ofradiation. After having recovery time is provided for no longer than 24hours to the mice irradiated with 10 Gy of radiation, a selected peptidephage sample was injected thereto, and in vivo imaging are examineduntil the 2^(nd) day of the injection.

FIG. 8 shows results verifying specific binding ability of a peptidesequence discovered in each population through immunohistochemistry.

FIG. 9 shows preparation of a liposome, a drug encapsulation process andoptimization thereof. As a result of verifying a liposome manufacturingprocess, a drug encapsulation process, and size distribution of drug, itis confirmed that there is no difference in size before and after drugencapsulation and that drugs are evenly distributed.

FIG. 10 shows results of linking a peptide to a liposome including to aliposome including a drug encapsulated therein. FIG. 10A is a schematicdiagram showing linking of a peptide to a liposome including a drugencapsulated therein and also shows a chemical constitutional formularepresenting actually linked residues, and FIG. 10B shows results of areduction test to calculate the number of —SH residues in a liposomebefore being linked to a peptide and also shows results confirmingstability through verification of the size distribution after beinglinked to a peptide.

FIG. 11 shows in vivo imaging results for verifying targeting ability ofa material in which a peptide is linked to a liposome including a drugencapsulated therein. It is confirmed that only a liposome linked to apeptide is targeted in an irradiated mouse.

FIG. 12 shows in vivo tumor growth delay results for verifyingpossibility of a material in which a peptide is linked to a liposomeincluding a drug encapsulated therein to be used as an anticancer drug.In this regard, only a liposome linked to a peptide and including a drugencapsulated therein is proved to be effective in treating tumors in anirradiated group.

FIG. 13 shows results confirming targeting ability of a selected peptideupon irradiation to mice transplanted with other patient's gastriccancer tissue according to Example 8 of the present invention.

BEST MODE

The present invention provides a peptide for targeting gastric cancer,the peptide including an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1 TO 6. The above-mentioned amino acidsequences are shown in Table 1.

The peptide of the present invention is a low-molecular weight peptideconsisting of 7 amino acids. Such a low-molecular weight peptide issmall in size so that it can be stabilized three-dimensionally. Inaddition, a low-molecular weight peptide has the advantage of being ableto easily pass through a membrane and to recognize a target moleculedeep in tissues. Since the stability of the low-molecular weight peptideof the present invention is secured through local injection and theimmunoreactivity can be minimized, there is an advantage that cancer canbe diagnosed early. In addition, the mass production of thelow-molecular weight peptide of the present invention is relatively easycompared that of an antibody, and the toxicity of the low-molecularweight peptide of the present invention is weak.

In addition, the low-molecular weight peptide of the present inventionis has an advantage of a strong binding force to a target materialcompared to an antibody, and do not undergo denaturation duringthermal/chemical treatment. In addition, due to a small molecular size,the low-molecular weight peptide can be used as a fused protein as beingattached to other proteins. In detail, the low-molecular weight peptidecan be also used as being attached to a high-molecular weight proteinchain, and accordingly, can be used as a diagnosis kit and a drugdelivery carrier.

The low-molecular weight peptide of the present invention can be easilyprepared according to the chemical process known in the art (Creighton,Proteins; Structures and Molecular Principles, W. H. Freeman and Co.,NY, 1983). As representative methods, liquid or solid phase synthesis,fractional condensation, F-MOC or T-BOC chemical method, or the like maybe used (Chemical Approaches to the Synthesis of Peptides and Proteins,Williams et al., Eds., CRC Press, Boca Raton Fla., 1997; A PracticalApproach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989),but the method is not limited thereto.

In addition, the low-molecular weight peptide of the present inventioncan be prepared according to a genetic engineering method. First,according to a conventional method, a DNA sequence encoding the sequencelow-molecular weight peptide is prepared. Here, a DNA sequence can beprepared by PCR amplification using an appropriate primer.Alternatively, according to a standard method known in the art, a DNAsequence can be synthesized using, for example, an automatic DNAsynthesizer (manufactured by Biosearch or AppliedBiosystems). Such asynthesized DNA sequence is inserted to a vector including one or moreexpression control sequences (for example: a promoter, an enhancer, orthe like) that are operatively linked with the DNA sequence to controlexpression of the DNA sequence, and then, a host cell is transformedwith a recombinant expression vector prepared therefrom. A resultingtransformant is cultured in an appropriate medium under suitableconditions to allow the expression of the DNA sequence, so thatsubstantially pure peptides that are encoded by the DAN sequence arerecovered from the culture. Such recovery may be performed according toa method known in the art (for example, chromatography). The term‘substantially pure peptides’ used herein refers to peptides that do notsubstantially include any protein derived from the host.

In addition, the present invention provides a peptide for targetinggastric cancer, the peptide being specific to an irradiated gastriccancer tissue and including an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1 to 3.

In detail, a peptide including an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1 to 3 specifically binds to a gastriccancer tissue, in particular, an irradiated gastric cancer tissue.

The term “target” or “specific” used herein refers to ability tospecifically bind only to a gastric cancer tissue, especially anirradiated gastric cancer tissue, without binding to other normaltissues. A gastric cancer-specific peptide can specifically bind to theinside or outside of a gastric cancer tissue.

In addition, the present invention provides a polynucleotide encoding anamino acid sequence selected from the group consisting of SEQ ID NOs: 1to 3.

The term “polynucleotide” used herein refers to a single-stranded ordouble-stranded polymer of deoxyribonucleotides or ribonucleotides. Sucha polynucleotide includes a RNA genome sequence, a DNA sequence (forexample, gDNA and cDNA), and a RNA sequence transcribed from the DNAsequence. Unless otherwise mentioned, a polynucleotide includes ananalog of a natural polynucleotide.

The polynucleotide includes not only a nucleotide sequence that encodesthe peptide for targeting gastric cancer, but also a sequencecomplementary to the nucleotide sequence, wherein such a complementarysequence includes not only a perfectly complementary sequence, but alsoa substantially complementary sequence.

In addition, the polynucleotide may be subjected to modifications. Suchmodifications include addition, deletion, non-conservative substitution,or conservative substitution of a nucleotide. The polynucleotideencoding the amino acid sequence is also interpreted to include anucleotide sequence that exhibits substantial identity to the nucleotidesequence. The substantial identity is obtained by aligning thenucleotide sequence with any other sequences to the greatest extent andby analyzing the aligned sequence using algorithms commonly used in theart, and in this regard, the substantial identity may indicate asequence having at least 80% homology, at least 90% homology, or atleast 95% homology with the aligned sequence.

In addition, the present invention provides a composition for diagnosinggastric cancer, the composition including a peptide including an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.

In addition, the present invention provides a composition forradio-sensitive diagnosing gastric cancer, the composition including apeptide being specific to an irradiated gastric cancer tissue andincluding an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1 to 3.

The term “diagnosis” used herein refers to identification of thepresence or characteristic of a pathological condition. For the purposeof the present invention, the diagnosis is to identify the presence orcharacteristic of gastric cancer.

The diagnosis of gastric cancer using the peptide of the presentinvention may be performed by detecting binding of the peptide of thepresent invention to a corresponding tissue or cell directly obtainedfrom blood, urine, or biopsy.

addition, to easily confirm, detect, and quantify binding of the peptideof the present invention to the gastric cancer tissue, the peptide ofthe present invention can be provided in a labeled state. That is, thepeptide provided herein may be linked to a detectable label (forexample, via covalent binding or cross-linking). The detectable labelmay be a chromogenic enzyme (for example, peroxidase and alkalinephosphatase), a radioactive isotope (for example, ¹²⁴I, ¹²⁵I, ¹¹¹In,⁹⁹mTc, ³²P, and ³⁵S), a chromophore, or a luminescent material or afluorescent material (for example, FITC, RITC, rhodamine, cyanine, TexasRed, fluorescein, phycoerythrin, or quantum dots).

Similarly, the detectable label may be an antibody-epitope, a substrate,a cofactor, an inhibitor, or a affinity ligand. Such labeling may beperformed during the synthesis of the peptide of the present invention,or may be additionally performed on a peptide that is alreadysynthesized. When using a fluorescent material is used as a detectablelabel, cancer may be diagnosed according to fluorescence mediatedtomography (FMT). For example, the peptide of the present inventionlabeled with a fluorescent material may be circulated into the blood,and the fluorescence by the peptide may be observed by FMT. Iffluorescent is observed, it is diagnosed as cancer.

In addition, the present invention provides a composition for deliveringa drug, the composition including the peptide for targeting gastriccancer.

The peptide of the present invention may be used as an intelligent drugdelivery vehicle that selectively delivers a drug to a cancer tissue. Ifthe peptide of the present invention is used in combination with drugsof the related art in terms of treatment of cancer, the peptide of thepresent invention selectively delivers a drug only to a cancer tissueand a cancer cell, so that drug efficacy may be increased while drugside effects on a normal tissue may be significantly reduced at the sametime.

For use as the drug, any anticancer drug that is conventionally used inthe treatment of cancer can be used so long as the anticancer drug isable to be linked to the peptide of the present invention. Examples ofthe drug include cisplatin, 5-fluorouracil, adriamycin, methotrexate,vinblastine, busulfan, chlorambucil, cyclophosphamide, melphalan,nitrogen mustard, nitreosourea, taxol, paclitaxel, docetaxel,6-mercapropurine, 6-thioguanine, bleomycin, daunorubicin, doxorubicin,epirubicin, idarubicin, mitomycin-C, and hydroxyurea. In addition, thelinking of the anticancer drug to the peptide of the present inventionmay be performed by a method known in the art, for example, covalentbonding, cross linking, or the like. For this purpose, the peptide ofthe present invention may be, if necessary, subjected to chemicalmodifications to the extent that the activity thereof is not lost.

MODE OF THE INVENTION

Hereinafter, to promote understanding of one or more exemplaryembodiments, reference has been made in detail to embodiments. Thepresent invention, however, may be embodied in many different forms andshould not be construed as being limited to the exemplary embodimentsset forth herein. Rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the present invention to one of ordinary skill in the art.

<Example 1> Establishment of a Mouse Model Transplanted with a Patient'sGastric Cancer Tissue

Considering overcoming limitations that existing animal modelstransplanted with cultured cancer cells had, an ideal animal model ofcancer similar to actual patient's cancer microenvironments is prepared.Then, to establish an animal model that can confirm influence of anirradiation-dependent cancer tissue, first, a mouse model transplantedwith a cancer tissue that was extracted from an actual patient havinggastric cancer was established. Regarding the establishment of such ananimal model, the cancer tissue extracted from a patient was cultured inan NOD/SCID mouse. Once the cancer tissue was found in the NOD/SCIDmouse, subculturing was carried out by using a Balb/c nude mouse,beginning from the next subculturing. In detail, after an NOD/SCID mousewas anesthetized via intraperitoneal injection of anesthetics, thecancer tissue was cut into pieces each having a size of 3×3 mm. Next,each of both flanks of the NOD/SCID mouse was transplanted with a pieceof the cut cancer tissues, treated with a mixed solution of 100 μl of100 IU/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, and2.5 μg/ml amphotericin B antibiotics, and then, sutured. The NOD/SCIDmouse was recovered on a heating pad (for about 2 hours). Afterwards,the formation and growth of tumors were observed every week. When thecancer tissues grew to a size of 400-500 mm³, the cancer tissues wereseparated and cut into pieces each having a size of 3×3 mm forsubculturing. Next, for next subculturing, a nude mouse was anesthetizedvia intraperitoneal injection of anesthetics, and a piece of the cutcancer tissues was transplanted subcutaneously on the right thigh of thenude mouse. A transplantation site was changed from the flank to theboth thighs so that irradiation can be locally done without affectingother organs during irradiation. That is, a mouse model in which acancer tissue that underwent subculturing and was re-transplanted up tofour times was formed was established. The growth of the cancer tissuewas observed for about a month (4 weeks), and when the size of thecancer tissue was increased to about 200 mm³, only the cancer tissue waslocally irradiated with 10 grays (Gy) of radiation. After havingrecovery time for no longer than 24 hours, in vivo peptide screening wasperformed. Here, as a control group in an irradiated population, a mousemodel that was not irradiated among the same mouse models was used toscreen a peptide. The results of the establishment of such a mouse modelare shown in FIG. 1.

<Example 2> In Vivo Screening of M13 Phage Peptide Library—In Vivo PhageDisplay

Regarding the mouse model established according to Example 1, i.e., aradio-sensitive xenograft mouse model transplanted with a patient'sgastric cancer tissue, a method for identifying a peptide having highspecificity during in vivo peptide screening using a random loop peptidelibrary was designed. For use as the library, a loop peptide librarymanufactured to have about 2.7 billion different amino acids sequencesvia random array of 7 amino acids [(i.e., a library fused with an M13phage gp3 minor coat protein)-Ph.D™ phage display peptide library kit,New England Biolabs (NEB)] was purchased. To screen a peptide showingspecific binding to the irradiated gastric cancer tissue in theestablished mouse model, a M13 phage peptide library (i.e., a libraryfused with an M13 phage gp3 minor coat protein and consisting of 7 aminoacids having about 2.7 billion different amino acids sequences) wasinjected into the tail vein of the mouse so that e M13 phage peptidelibrary was circulated in vivo for 15 minutes (also known as a method ofbinding an in vivo cancer tissue with an M13 phage peptide library).Then, during this process, a peptide-expressing phage specificallybinding to the cancer tissue was selected with different washingconditions. Such a screening scheme is shown in FIG. 2. In detail, FIG.2 an M13 phage screening scheme for screening a cancer tissue-targetingpeptide, wherein (1) a mouse model in which a cancer tissue was formedon the right hind leg was established, (2) a phage library in which aloop peptide library consisting of 7 amino acids was expressed on asurface of a M13 phage was injected into the tail vein of the mouse toallow circulation of the phage library, (3) phages were washed under avariety of washing conditions, and (4) phages were obtained by elutingfinally targeted phages. The eluted phages infected Escherichia coli,and were injected again into the tail vein of the mouse to allowcirculation of the phages. By repeating such cycles under washingconditions with high intensity, a process of screening phages havinghigh specificity and a strong binding strength was repeatedly performed(also known as biopanning). Biopanning was performed five times percycle so that a phage expressing a sequence of a peptide specificallybinding to the patient's in vivo gastric cancer tissue was obtained. Toconfirm whether the peptide-expressing phage actually targeted thecancer tissue only, other in vivo organs were also subjected tocomparison. That is, for every biopanning, phages were eluted from eachof extracted heart, lung, liver, spleen, kidney, and cancer tissue, andthe phage concentration was measured for comparison. The results of thecomparison are shown in FIG. 3. Finally, the obtained phages infected E.coli ER2738 cells that are host cells, and were subjected toamplification in an LB medium. Then, 100 phage plaques were selectedrandomly from each of an irradiated population (experimental group) anda non-irradiated population (control group), and the M13 phage genomicDNA (single-stranded circular DNA) was separated and purified toidentify a gene sequence, thereby identifying an amino acid sequence ofa peptide expressed in a phage-surface protein (e.g., a gp3 minor coatprotein) and targeting the cancer tissue. The results of theidentification are shown in Table 1. Table 1 shows a summary ofsequences discovered from each of the irradiated (experimental group)and the non-irradiated population (control group) by using the Clustal Xprogram for sequence analysis.

TABLE 1 Group No. Peptide sequence Irradiated P1 TVRTSAD (SEQ ID NO: 1)population (10  P2 RYVGTLF (SEQ ID NO: 2) Gy) P3 NRGDRIL (SEQ ID NO: 3)Non-irradiated W1 NWGDRIL (SEQ ID NO: 4) population as W2QRSLPSL (SEQ ID NO: 5) control W3 DVWHSAY (SEQ ID NO: 6)

<Example 3> In Vivo Imaging for Confirming Targeting Ability of a LoopPeptide-Expressing Phage Regarding a Patient's Gastric Cancer Tissue

To verify, based on in vivo imaging, whether the obtained phageexpressing a loop peptide has exhibited specific binding abilityfollowing amplification and to confirm targeting efficiency of theobtained phage, a process of linking a fluorescent probe was performedfirst. In particular, 1 μg/μl of N-hydroxysuccinimide esters of Cy5.5(Amersham) was added to 1 mL of bicarbonate buffer (pH 8.3) having thephage concentration of 10¹¹ plaque forming units (pfu), and then, in acondition where a dark environment was maintained, 3 a phage-surfaceprotein was linked to the Cy5.5 fluorescent probe at room temperaturefor 3 hours. That is, loop peptide-expressing phages to which the Cy5.5fluorescent probe was linked were each obtained by precipitation with170 μl of 20% (w/v) PEG 8000/2.5 M NaCl solution and purification. Todetermine a proportion of the Cy5.5 fluorescent probe linked to each ofthe finally obtained phage samples, an IVIS spectrum imaging system(Xenogen) was used for measurement, and region of interest (ROI) valueswere determined using the software program of a corresponding device.Accordingly, it was confirmed that the Cy5.5 fluorescent probe waslinked to each of the phage samples at almost the same linkingproportion. The corresponding results above are shown in FIG. 4.

After each of the prepared phages expressing the loop peptide wasinjected into the ratio-sensitive xenograft mouse model of Example 1 andthe control group through the vein tail of the mouse, images weremeasured for 2 days immediately after the injection, thereby confirmingimages showing in vivo circulation of the peptide and the targeting ofthe peptide only in the cancer tissue while the targeting to otherorgans and tissues gradually disappeared. In this regard, the peptidewas proved to completely target the in vivo gastric cancer tissue. Inaddition, the excellent targeting ability of the peptide sequence thatwas identified by biopanning according to Example 2 was resulted fromthe in vivo imaging and showed in FIG. 5. In addition, to verify whichpart of the cancer tissue was targeted by each of the looppeptide-expressing phages via ex vivo imaging, only the cancer tissuewas separated and extracted, and the whole cancer tissue itself and therespective cancer tissue were subjected to fragmentation into severalpieces. The imaging results obtained therefrom are shown in FIG. 6.

To compare targeting efficiency more accurately based on the results ofFIGS. 5 and 6, the pieces of the extracted cancer tissue the pieces ofthe extracted cancer tissue were collected independently, and phagesbound to the cancer tissues were eluted. The concentration of each ofthe eluted phages was measured according to titering, and due todifferent size and weight of the extracted cancer tissue, the weight ofeach cancer tissue was also measured in terms of establishing numericalstandardization. In this regard, the amount of the identified phages wascalculated relative to the weight of the cancer tissue. In addition, tomore accurately quantify each imaging result, the in vivo imaging andthe ex vivo imaging were confirmed by measuring ROI values that weredetermined using the IVIS spectrum (Xenogen) program, and the resultsthereof are shown in Table 2.

TABLE 2 Group Sample Sequence pfu/mg in vivo ROI ex vivo ROI IrradiatedCy5.5 — —  1.00  1.00 population (10 Empty — —  7.37 24.58 Gy) P1TVRTSAD 25.3 16.44 70.66 P2 RYVGTLF  6.3  9.05 38.83 P3 NRGDRIL 18.619.83 77.85 Non-irradiated Cy5.5 — —  1.00  1.00 population as Empty — — 0.95  0.54 control W1 NWGDRIL 11.8  1.86  2.08 W2 QRSLPSL 18.2  2.19 2.68 W3 DVWHSAY  8.1  1.90  3.54

<Example 4> In Vivo Imaging for Confirming Selectively Binding PeptideSequence Upon Irradiation

To confirm whether 3 peptide sequences identified in Example 3 wereresponsive to cancer microenvironments during irradiation, the targetingefficiency of these irradiation-dependent peptide sequences wasconfirmed. In detail, in the presence of differences only in irradiationin the same mouse model transplanted with the patient's gastric cancer,targeting of the peptide which was dependent upon cancermicroenvironments was subjected to verification. Accordingly, as in themouse model established in Example 1, mice in which tumor was formed bytransplantation of a patient's gastric cancer tissue were selected.Among the selected mice, only some of them were irradiated to therebyestablish a control group and an experimental group. In the same manneras in Example 3, the selected phages expressing the peptide wereamplified and fluorescent labeling was also performed thereon, The samesample was injected into an irradiated mouse group and a control groupthereof, thereby obtaining images for the last two days. Consequently,when comparing targeting in the irradiated mouse group with that in thecontrol group, the two groups showed differences in the targetingefficiency. The imaging measurement results of the present embodimentare shown in FIG. 7. As shown in FIG. 7, the control group including themouse model transplanted with the patient's gastric cancer tissue showedlow targeting efficiency, whereas the irradiated mouse model includingthe same mouse model transplanted with the patient's gastric cancertissue showed specific binding ability through images.

<Example 5> Histological Verification of Selective Binding Ability of aDiscovered Peptide

To verify histological targeting ability of the selected peptide (threesequences per population), each population was injected via the tailvein. After 24 hours, cancer tissues of each population were extractedto prepare paraffin blocks and slices. In detail, (1) the extractedcancer tissues were immersed in a formaldehyde solution at roomtemperature for 24 hours in terms of for immobilization. Then, followinga dehydration process, paraffin was added to the solution throughpenetration to form paraffin blocks. Afterwards, microtome was used tomanufacture slices having a thickness of 3 μm. To perform realimmunohistological staining, (2) following a deparaffinization processperformed on the slices, (3) an unmasking process was performed so thatstructures of various proteins immobilized to the tissue slices wererecovered to restore sites where antibodies normally bind. Sequentially,(4) a blocking process was performed using a 5% BSA solution, primaryantibodies were bound (wherein the antibodies used herein wereanti-mouse M13 IgG recognizing M13 phage capsid proteins), (6) andsecondary antibodies were bound while HRP was bound. Afterwards, (7)sites where phages were present were stained using DAB development, (7)the nuclei of the phages were stained with hematoxylin, and (8) adehydration process was performed thereon. Once completed, mounting wasperformed so that the tissue slices that were immunohistochemicallystained were permanently preserved. The results of immunohistochemicalstaining performed as described above are shown in FIG. 8.

<Example 6> Liposome Preparation, Drug Encapsulation, andPeptide-Liposome Linking Process

In detail, five lipids constituting a liposome, such asdipalmitoylphosphatidylcholine (DPPC, concentration of 50 mM),dipalmitoylphosphatidylglycerol (DPPG-Na, concentration of 50 mM),N-[3-(2pyridinyldithio)-1-oxopropyl]-L-α-dipalmitoylphosphatidylcholine(DPPE-PDP, concentration of 50 mM), cholesterol (concentration of 200mM), and cholesterol—PEG (concentration of 200 mM), were each dissolvedin an organic solvent containing methanol and chloroform (prepared at aratio of 1:1). DPPG-Na which does not melt at room temperature wascompletely dissolved at 55° C. for more than 30 minutes. Each of thefive dissolved lipids was added to a round-bottom flask so as to preparea mixed solution containingDPPC:DPPE-PDP:DPPG-Na:cholesterol-PEG:cholesterol at a ratio of15:15:30:4:36. The round-bottom flask was rotated at 55° C., and waspressurized for about 2 to 3 hours to volatilize the organic solventtherefrom. Meanwhile, a thin lipid film was formed within theround-bottom flask. When a white thin film was formed within theround-bottom flask, the organic solvent remained at room temperature wascompletely volatilized, so that only a pure lipid film remained.Afterwards, to prepare a liposome using the pure lipid film, 3 ml ofHEPES (10 nM, pH 4) buffer was added thereto, and the round-bottom flaskwas rotated in a thermostat (55° C.) for 1 hour to dissolve the purelipid film. To dissolve it sufficiently, a vortex was used to stronglyshake the round-bottom flask, so that the pure lipid film was able to becompletely dissolved without leaving any agglomerate. To make the sizeof the prepared liposome uniform, a nitrogen gas extruder and apoly-carbonate filter were used to filter the liposome through a filterwith fine holes, wherein the fine holes used herein had a diameter of800 nm, 400 nm, 200 nm, and 100 nm in the stated order. Consideringaccurate size and uniformity of the liposome, a filter having a diameterof 200 nm and a filter having a diameter of 100 nm were used twice orseveral times for extrusion. To load a drug into the extruded liposome,the buffer containing the liposome dissolved therein was replaced withPBS (pH 7.0) using a Sephadex column. Accordingly, the resultingliposome was dissolved in the buffer such that the inside of theliposome had pH 4.0 and outside thereof had pH 7.0. The liposomeresulting from the completion of buffer replacement and doxorubicindissolved in buffer having pH 7.0 were mixed in a round-bottom flask.Then, to encapsulate the drug dissolved in the buffer having pH 7.0within the liposome having pH 4.0 by a concentration gradient, theround-bottom flask was rotated in a bath at 60° C. for 20 minutes. Then,to isolate the remaining non-encapsulated drug, a pure liposomeincluding the drug encapsulated therein was purified using a Sephadexcolumn. According to the Dinamic light scattering; DLS method, the drugdelivery carrier was optimized by measuring the size and stability ofthe finally prepared drug-encapsulated liposome. The results of the drugdelivery process and optimization thereof described above are shown inFIG. 9.

Among the peptides verified in Example 5, the peptide having ‘TVRTSAD’sequence was synthesized by a request, and ligated with thedrug-encapsulated liposome of Example 6. Accordingly, a peptide in whichthe C-terminal of the ‘TVRTSAD’ was linked with a Cy7 fluorescent probeand the N-terminal of the ‘TVRTSAD’ was free from any process to belinked with a liposome was prepared by a request from AnyGen Inc. (SouthKorea). The residue of the N-terminal of the prepared peptide wasprocessed to be linked with a thiol group (—SH) of a liposome via adisulfide bond. Before performing linking with the liposome, the numberof the thiol group of the liposome was counted, and a liposomalreduction test was conducted so as to link the liposome to the peptidedepending on the ratio of the thiol group. DTT 1 mM was added andpyridine 2-thione was measured at OD₃₄₃ nm, to count the number of thethiol group of the liposome. Afterwards, to link the liposome with thepeptide, the liposome and the peptide were mixed at a molar ratio of1:1.5 to allow a reaction for 2 hours at room temperature. Then, toisolate unreacted (unlinked) peptide, the liposome linked with a purepeptide was purified using a Sephadex column. Afterwards, to verify thatthere is no change in the size and stability of the liposome before andafter being linked with the peptide, the verification was demonstratedaccording to the Dinamic light scattering; DLS method, and the resultsare shown in FIG. 10.

<Example 7> Verification of Targeting Ability of Peptide-Linked Liposomeand Validation of New Concept Anticancer Drug

To verify whether the drug carrier of Example 6 in which the ‘TVRTSAD’sequence was linked to the drug-encapsulated liposome actually played afunction in the living body, in vivo imaging was performed. In detail,the radio-sensitive xenograft mouse model of Example 1 and the controlgroup were each injected through the vein tail of the mouse, and imageswere measured for 2 days immediately after the injection, therebyconfirming that images showing in vivo circulation of the peptide andthe targeting of the peptide only in the cancer tissue while thetargeting to other organs and tissues gradually disappeared. In thisregard, the peptide was proved to completely target the in vivo gastriccancer tissue. The results of the in vivo imaging are shown in FIG. 11.

In addition to the in vivo imaging, in vivo tumor growth delay was alsoperformed to validate the peptide as a target drug delivery carrier. Theradio-sensitive xenograft mouse model of Example 1 and the control groupwere established, and once the tumor size was increased to about 100mm³, grouping was performed thereon. In this regard, a total of 5groups, i.e., 1; PBS, 2; irradiation (2 Gy), 3; DOX(2 mg/kg)+irradiation(2 Gy), 4; LP-DOX (2 mg/kg)+irradiation (2 Gy), 5; P1(peptide)-LP-DOX (2mg/kg)+irradiation (2 Gy), were prepared. Here, test group wasdesignated as Group 5 while the control groups were designated as Groups1 to 4 for observation. Each group included 5 mice (n=5). Compared tothe control groups, the test group (i.e., Group 5) showed that the tumorsize was significantly small, and accordingly, the results of validationof a new concept anticancer drug are shown in FIG. 12.

As verified in FIGS. 11 and 12, the material linked with thecorresponding peptide and the drug-encapsulated liposome were verifiedto be utilized in the in vivo imaging and the in vivo tumor growthdelay, and accordingly, the possibility of the material as a new conceptanticancer drug that can simultaneously diagnose and treat cancer wasproved.

<Example 8> In Vivo Imaging for Verifying Selective Binding Ability of aPeptide Upon Irradiation on a Gastric Cancer Tissue of Other Patients

To verify whether the peptide having selective binding ability uponirradiation on the patient's gastric cancer tissue obtained in Examplesabove also exhibited the same selective binding ability in casesassociated with gastric cancer tissues of other patients having the samegastric cancer and the same characteristics upon irradiation, other thanthe corresponding gastric cancer case showing selective binding of thepeptide upon irradiation, gastric cancer tissues each extracted fromdifferent patients were prepared to establish a mouse model. In detail,in addition to the patient's gastric cancer tissue used in Exampleabove, two gastric cancer tissues of other patients were prepared,wherein all the gastric cancer tissues used herein were characterized asadenocarcinoma. In the same manner as in Example 1, mouse modelsincluding each of the corresponding gastric cancer tissues wasestablished, and some of them were irradiated to thereby establish acontrol group and an experimental group. According to the in vivoimaging which is the same method as the one used for confirmingtargeting efficiency in Example 4, the irradiation-dependent targetingefficiency of the peptide was verified. The amplification of phagesexpressing the selected peptide and the fluorescent labeling wereperformed in the same manner as in Example 3. Then, the completed samplewas injected into each of the irradiated test mouse group and thecontrol group, and images thereof were confirmed on the 2^(nd) day ofthe observation. As a result, it was confirmed that the two gastriccancer tissues of other patients also showed selective accumulation ofpeptide-phage only in the tumors of the irradiated test mouse group, inthe same manner as in the existing gastric cancer tissue of the patient.The results of the imaging measurement of the corresponding embodimentsare shown in FIG. 13. As shown in FIG. 13, it was confirmed that thepeptide-expressing phage did not target the cancer tissue when there isno irradiation applied to the cancer tissue, whereas thepeptide-expressing phage targeted the cancer tissue when irradiation isapplied to the cancer tissue. In conclusion, it was confirmed that thepeptide sequence selected in the corresponding technology was clearlyverified as the peptide sequence selectively targeting the gastriccancer upon irradiation, and that the targeting ability of thecorresponding peptide is not limited to the patient's gastric cancertissue only. That is, as the peptide sequence exhibiting selectivetargeting ability only in the case where the gastric cancer tissue ofother patients are irradiated, it was verified that the scope ofapplication of the peptide of the present invention is not limited inclinical applications.

It should be understood that embodiments described herein should beconsidered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments. While one or more embodiments have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope asdefined by the following claims.

1. A peptide for targeting gastric cancer, the peptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 1to
 6. 2. The peptide of claim 1, wherein the peptide comprising an aminoacid selected from the group consisting of SEQ ID NOs: 1 to 3 isspecific to an irradiated gastric cancer tissue.
 3. A polynucleotideencoding the peptide of claim
 1. 4. A method for diagnosing gastriccancer, comprising: obtaining a gastric cancer tissue sample from apatient; transplanting the tissue sample into a subject; applying thecomposition comprising the peptide of claim 1 to the subject; andidentifying the presence of the gastric cancer.
 5. A method fordiagnosing radio-sensitive gastric cancer, comprising: obtaining agastric cancer tissue sample from a patient; transplanting the tissuesample into a subject; applying the composition comprising the peptideof claim of 2 to the subject; and identifying the presence of theradio-sensitive gastric cancer.
 6. The method of claim 4, wherein thepeptide is labeled with one selected from the group consisting of achromogenic enzyme, a radioactive isotope, a chromopore, and aluminescent or fluorescent material.
 7. A composition for delivering adrug, the composition comprising the peptide of claim
 1. 8. Thecomposition of claim 7, wherein the drug comprises an anticancer drug.